In vitro fertilization (IVF) is a process by which an egg is fertilized by sperm outside the body. In vitro IVF is a major treatment for infertility when other methods of assisted reproductive technology have failed. The process involves monitoring a woman's ovulatory process, removing egg or eggs from the woman's ovaries and letting sperm fertilize them in a fluid medium in a laboratory. When a woman's natural cycle is monitored to collect a naturally selected ovum (egg) for fertilization, it is known as natural cycle IVF. The fertilized egg (zygote) is then transferred to the patient's uterus with the intention of establishing a successful pregnancy. The first successful birth of a “test tube baby”, Louise Brown, occurred in 1978. Louise Brown was born as a result of natural cycle IVF. Robert G. Edwards, the physiologist who developed the treatment, was awarded the Nobel Prize in Physiology or Medicine in 2010. (S K Kalra et al. Fertility and Sterility Vol 95, no 6 p 1888-1889. In vitro fertilization and adverse Childhood outcomes: what we know, where we are going, and how we will get there. A glimpse into what lies behind and beckons ahead. B Almog et al. Fertility and Sterility Vol 94, No 6 p 2026-2028. Promoting implantation by local injury to the endometrium).
In spite of dramatic progress in assisted reproduction technology over the past 25 years, the overall effectiveness of even the most advanced treatments such as IVF/embryo transfer (IVF/ET) is relatively low, averaging at about 20-30% live births per treatment cycle (Nyboe-Andersen et al., Hum Reprod. 2009; 24(6):1267-87).
Embryo transfer is an independent factor affecting the outcome of the treatment The determinants of success of embryo transfer involve the quality of embryo(s), uterine receptivity and the quality of the intrauterine environment (Cavagna and Mantese, Placenta. 2003; 24 Suppl B:S39-47).
Uterine contractions constitute the most fundamental components of uterine receptivity. Contractile activity of the uterus plays an important role in embryo implantation. Excessive uterine contractions may decrease implantation rates in IVF cycles as contractile activity might expel embryos from the uterus (Fanchin et al. Hum Reprod, 1998; 13: 1968-74). Up to date, treatment strategies to reduce uterine contractions before embryo transfer such as the use of beta agonists or non-steroid anti-inflammatory drugs have not been shown to provide sufficient benefit; Moon et al., Fertil Steril 2004; 82:816-20; Tsirigotis et al. Human Reproduction and Embryology, Jun. 25-28, 2000; Bologna, Italy:).
Treatment cycles induce an abundant increase in oestradiol concentrations which are about 10-20 nmol/l at the end of ovarian stimulation as compared with less than 2 nmol/l before the ovulation in the natural cycle Supraphysiological concentrations of oestradiol are expected to induce local (endometrial) production of oxytocin, formation of oxytocin receptors, and—indirectly—formation/release of PGF2a which is in fact similar to the prelabour status. Also, both oxytocin and vasopressin are involved in induction and maintenance of uterine contractions during labour
It has been shown that uterine contractile activity in IVF cycles is increased by approximately 6-fold when measured before embryo transfer as compared with the situation before ovulation in the natural cycle. Uterine contractions play an important role in human reproduction, being actively involved in rapid and directed sperm transport and high fundal embryo implantation
In IVF/ET treatments, a progressive decrease in uterine contractions is observed after the egg collection, reaching nearly a quiescent status at the time of blastocyst transfer (5-6 days after egg collection) (Fanchin et al., Fertil Steril 2001; 75: 1136-40). Such a decrease in contractile activity is thought to further augment the higher implantation rates achieved with blastocyst transfers. However, the majority of embryos are still transferred on day 2 or 3 after fertilization, during periods of noticeable uterine contractile activity.
The embryo transfer procedure itself is expected to increase the local oxytocin and prostaglandins release Any additional manipulation of the vagina or cervix, such as the use of a tenaculum, provides an additional stimulus for oxytocin/prostaglandin release (Dorn et al., 1999), which is coupled with increases in uterine contractions). Mansour et al. demonstrated that, in more than half of patients having mock embryo transfer with methylene blue dye, the dye was seen to be transported into the vagina after the procedure (Mansour et al., Hum Reprod 1994; 9: 1257-9). It was also demonstrated that less than 50% of transferred embryos remained in the uterus 1 h after transfer and about 15% of embryos could be found in the vagina after embryo transfer (Poindexteret al. Fertil Steril, 1986; 46:262-7).
Considering the above, it has been suggested that uterine contractile activity at the time of embryo transfer and especially fundo-cervical contractions could expel embryos from the uterus Fanchin et al. Human Reprod 1998; 13:1968-74 have estimated that about 30% of patients undergoing embryo transfer have pronounced uterine contractions. In that group, success rates of IVF/ET treatment were up to 3-fold less compared with the population of patients with ‘silent’ uteri (16% versus 53% of clinical pregnancies). The difference was independent of the direction of uterine contractions noted during the assessments. That could imply that pharmacological inhibition of increased contractions at the time of embryo transfer could be an attractive target for potential treatment.
Interfering with the PGF2a/oxytocin systems and possibly improving endometrial perfusion could be one mechanism by which uterine contractions would decrease and improve uterine receptivity.
The effectiveness of in vitro fertilization-embryo transfer (IVF-ET) usually does not exceed 30% per treatment cycle (NyboeAndersen A, Gianaroli L, Felberbaum R, de Mouzon J, Nygren K. Assisted reproductive technology in Europe, 2001. Results generated from European registers by ESHRE. Hum Reprod 2005; 20:1158-76) and is further reduced in women older than 36 years. (Stolwijk A, Wetzels A, Braat D. Cumulative probability of achieving an ongoing pregnancy after in-vitro fertilization and intracytoplasmic sperm injection according to a woman's age, subfertility diagnosis and primary or secondary subfertility. Hum Reprod 2000; 15:203-9)
Good quality of embryos and optimal intrauterine environment are the basic determinants of success for ET, and the whole IVF-ET procedure. Ideal intrauterine conditions that enable implantation include appropriate endometrial status, sufficient endometrial perfusion and absence of excessive uterine contractions. In particular, increased uterine contractile activity may expel embryos from the uterus. (Mansour R, Aboulghar M A, Serour G I, Amin Y M. Dummy embryo transfer using methylene blue dye. Hum Reprod 1994; 9:1257-9) (Poindexter A, Thompson D, Gibbons W. Residual embryos in failed embryo transfer. Fertil Steril 1986; 46:262-7)
Implantation and pregnancy rates are inversely correlated with the frequency of uterine contractions and prostaglandin synthesis (PG synthesis). High uterine contractile activity at ET (five or more contractions per minute) is found in about one-third of patients, and in these women clinical pregnancy rates reach 13% per cycle, in contrast to the 53% of successful pregnancies in women with lower uterine activity (three or less contractions per minute) (Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13:1968-74.) Moreover, irritation of the uterine cervix by the ET catheter is likely to induce additional PG synthesis and contractile reflexes and further decrease the chances of successful embryo Implantation (Lesny P, Killick S, Tetlow R, Robinson J, Maguiness S. Embryo transfer—can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998; 13:1540-6.)
However, uterine contractile activity, an important component of uterine receptivity, is currently not a subject of specific diagnosis or treatment in ET recipients. Progesterone supplementation, even when acting on uterine receptivity, improving endometrial status, and decreasing uterine contractions, shows no benefit for pregnancy rates after IVF-ET. (Fanchin R, Righini C, de Ziegler D, Olivennes F, Ledee N, Frydman R. Effects of vaginal progesterone administration on uterine contractility at the time of embryo transfer. Fertil Steril 2001; 75:1136-40.)
Studies assessing the effectiveness of piroxicam (cyclooxygenase inhibitor) and ritodrine (β2-adrenoreceptor agonist) have shown a positive effect on pregnancy rates. (Moon H, Park S, Lee J, Kim K, Joo B. Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer. Fertil Steril 2004; 82:816-20; Tsirigotis M, Pelekanos M, Gilhespie S, Gregorakis S, Pistofidis G. Ritodrine use during the peri-implantation period reduces uterine contractility and improves implantation and pregnancy rates post-implantation. Presented at the 16th annual meeting of the European Society of Human Reproduction and Embryology; Jun. 25-28, 2000; Bologna, Italy). The drugs mentioned above have failed to enter routine clinical use because of safety concerns.
Recently, Moraloglu et al. Treatment with oxytocin antagonists before embryo transfer may increase implantation rates after IVF. Reproductive biomedicine online Sep. 2010; 21(3): 338-43 reported a randomized, placebo controlled trial with a total i.v. dose of 37.5 mg of atosiban (oxytocin antagonist) infused before and up to 2 h after the embryo transfer in 160 patients. The authors noted significant improvement in both implantation rates and clinical pregnancies. Implantation rates per embryo transferred were 20.4% versus 12.6% and clinical pregnancy rates per cycle were 46.7% versus 28.9% (atosiban versus placebo). Fewer early miscarriages were noted in the study group (16.7% versus 24.4%, atosiban versus placebo).
N-acetyl-L-cysteine (hereinafter referred to as NAC) is a well-known drug, which has been used mainly as a mucolytic agent and in the treatment of paracetamol poisoning. In recent years it has also been acknowledged as having other beneficial properties, such as being anti-inflammatory and anti-proliferative, and has been suggested for the treatment of a variety of different disorders and symptoms in addition to endometriosis also schizophrenia, diabetes and cancer.
To date, NAC has not been considered a drug in fertility treatment although it has been used for oral treatment of endometrioses as described in EP 2305238. In that study no conclusions about pregnancies were made. To the contrary when being treated for endometriosis it was not expected that any pregnancies could occur. Although, it has not been disclosed before, when analysing the data the inventors have noticed that there was one person who had taken NAC orally for endometriosis had a successful IVF treatment. Also two previous IVF trials had failed and resulted in an abortion. This was one of the reasons why the present inventors started investigating whether NAC could be used in connection with IVF.
Indeed, in mice, NAC was confirmed to support embryo implantation. In the animal model, embryo implantation rate was decreased when the mice were given oxytocin. NAC dose-dependently restored implantation rates in oxytocin-treated mice, which provided evidence for involvement of oxytocin in embryo implantation.